BAC 'landing' on chromosomes of Brachypodium distachyon for comparative genome alignment
BAC 'landing' on chromosomes of Brachypodium distachyon for comparative genome alignment
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DOI：
10.1038/nprot.2006.490
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发表时间：
2007-01-01
期刊：
影响因子：
14.8
通讯作者：
Hasterok, Robert
中科院分区：
文献类型：
--
作者：
Jenkins, Glyn;Hasterok, Robert
荧光原位杂交（FISH）使用细菌人工染色体（BAC）与大基因组DNA插入物作为探针（BAC“着陆”）是一种强有力的手段，通过该手段可以物理地绘制真核基因组图谱并进行比较。在这里，我们报告了一个BAC登陆协议，已专门为杂草物种二穗短柄草，这已通过最近由科学界作为一个替代模型的温带谷物和草。该协议描述了准备体细胞和减数分裂染色体底物FISH，BAC的标记，染色体定位策略，经验条件最佳原位杂交和严格洗涤，探针的检测和图像的捕获和处理。该方案的预期结果是将含有单拷贝插入片段的BAC特异性分配给该物种基因组的五个连锁群之一。一旦体细胞或减数分裂材料可用，整个方案可在约3d内完成。该协议已根据经验为B定制。二穗草及其近亲，但它可以适应不同的植物物种的微小修改。
Fluorescence in situ hybridization (FISH) using bacterial artificial chromosomes (BACs) with large genomic DNA inserts as probes (BAC 'landing') is a powerful means by which eukaryotic genomes can be physically mapped and compared. Here we report a BAC landing protocol that has been developed specifically for the weedy grass species Brachypodium distachyon, which has been adopted recently by the scientific community as an alternative model for the temperate cereals and grasses. The protocol describes the preparation of somatic and meiotic chromosome substrates for FISH, the labeling of BACs, a chromosome mapping strategy, empirical conditions for optimal in situ hybridization and stringency washing, the detection of probes and the capturing and processing of images. The expected outcome of the protocol is the specific assignment of BACs containing single-copy inserts to one of the five linkage groups of the genome of this species. Once somatic or meiotic material is available, the entire protocol can be completed in about 3 d. The protocol has been customized empirically for B. distachyon and its near relatives, but it can be adapted with minor modifications to diverse plant species.